Application Of α-L-arabinofuranidase From Bacillus Subtilis WB800N In The Hydrolysis Of Ginsenoside

Ginsenoside is the main active ingredient in ginseng,the content of glycosylated ginsenoside is higher in ginseng,while the content of decylated ginsenoside in ginseng is minimal or even non-existent,so it is called rare ginsenoside.However,compared with glycosylated ginsenosides,deglycosylated ginsenosides have higher pharmacological activities,such as anti-tumor,anti-coagulation,anti-injury sensation,heart protection,immune stimulation,memory enhancement and anti-depression activity,and have special effects in the pharmaceutical industry.At present,heat,acid treatment,fermentation,cell reaction and enzyme methods of conversion to transformation of ginseng saponin.Chemical treatment will cause pollution to the environment,high temperature treatment will produce side effects and reduce production.Due to the enzyme catalysis is environmental and do not have side effectsand,so it become the attention of the ginseng saponin conversion hot spots.The arabinofuran glycosidase can remove arabinofuran glycoside at the position of Rc C-20 of ginsenosides,also it can convert the glycosylated ginsenosides Rc into the rare ginsenosides Rd.In this paper,we cloned abfa and abf2 from Bacillus subtilis WB800N.Heterologous recombinant expression was performed in E.coli BL21（DE3）,purification,enzymatic property and ginsenoside transformation of the recombinant protein were studied.Through detection of ginsenoside transformation,both ABFA and ABF2 can transform ginsenoside Rc into ginsenoside Rd.ABFA and ABF2 were purified by Ni²⁺affinity chromatography,under optimum reaction conditions（pH 8.0,50℃;pH 8.0,60℃）,the two recombinant proteins have tolerance to glucose and arabinofuran sugar,and have a certain tolerance to most of the experimental metal ions and reagents,but not suitable for mixed reaction with ethanol.There are two glycoside hydrolase genes in our laboratory,glucosidase cbgl4,bifunctionalβ-xylanase/α-L-arabinopyranosidase xylarap51.Based on CBGL4 has enzymatic cleavage of Rb₁,Rb₂,Rb₃ C-3 external glucose,XYLARAP51 has enzymatic cleavage of Rb₂ and Rb₃ C-20 external xylose and arabinoglycosyl.By constructing the expression vector of Bacillus subtilis,the fusion gene was connected with the vector,so as to transform Rb₂,Rb₃ and Rc into F₂ by co-acting with arabinofuran glycosidases abfa and abf2 in Bacillus subtilis,thus constructing a complete transformation pathway.In this study,twoα-L-arabinofuran glycosidases capable of transforming ginsenoside Rc were extracted,and a recombinant protein was fused to express ginsenoside Rb₁ into Gyp XVII,Rb₂ and Rb₃ into Rd,laying a certain foundation for constructing a complete biological transformation pathway of ginsenoside F₂ in the later stage.